Cultured Rat Ventricular Myocytes Research Articles

Btg1 and Btg2 regulate neonatal cardiomyocyte cell cycle arrest

Rodent cardiomyocytes undergo mitotic arrest in the first postnatal week. Here, we investigate the role of transcriptional co-regulator Btg2 (B-cell translocation gene 2) and functionally-similar homolog Btg1 in postnatal cardiomyocyte cell cycling and maturation. Btg1 and Btg2 (Btg1/2) are expressed in neonatal C57BL/6 mouse left ventricles coincident with cardiomyocyte cell cycle arrest. Btg1/2 constitutive double knockout (DKO) mouse hearts exhibit increased pHH3+ mitotic cardiomyocytes compared to Wildtype at postnatal day (P)7, but not at P30. Similarly, neonatal AAV9-mediated Btg1/2 double knockdown (DKD) mouse hearts exhibit increased EdU+ mitotic cardiomyocytes compared to Scramble AAV9-shRNA controls at P7, but not at P14. In neonatal rat ventricular myocyte (NRVM) cultures, siRNA-mediated Btg1/2 single and double knockdown cohorts showed increased EdU+ cardiomyocytes compared to Scramble siRNA controls, without increase in binucleation or nuclear DNA content. RNAseq analyses of Btg1/2-depleted NRVMs support a role for Btg1/2 in inhibiting cell proliferation, and in modulating reactive oxygen species response pathways, implicated in neonatal cardiomyocyte cell cycle arrest. Together, these data identify Btg1 and Btg2 as novel contributing factors in mammalian cardiomyocyte cell cycle arrest after birth.

Lentiviral transduction of neonatal rat ventricular myocytes preserves ultrastructural features of genetically modified cells

Preserving morphological features that are important for cell function and structure is a critical parameter for in vitro experiments with rat cardiomyocytes. Lentiviral vectors are commonly used as gene transfer tool because of its high flexibility, efficiency to deliver expression cassettes and versatility of transducing quiescent cells. The tropism of the recombinant viral particle can be determined depending on the virus envelope, which shows a specific binding to cell surface receptors on the target cell. The combination of promoter arrangement and viral envelope must be optimized to achieve a greater transduction efficiency and a higher transgene expression. In this study we explored the optimization of promoters and heterologous envelopes to transduce primary culture of neonatal rat ventricular myocytes. Our results suggest a robust expression driven by the cytomegalovirus promoter, and high efficiency transduction mediated by VSV-G envelope with no apparent compromising ultrastructural features of genetically modified cells.

Electrically stimulable indium tin oxide plate for long-term in vitro cardiomyocyte culture

BackgroundWe investigated whether electrical stimulation via indium tin oxide (ITO) could enhance the in vitro culture of neonatal rat ventricular myocytes (NRVMs), which are important in vitro models for studying the mechanisms underlying many aspects of cardiology.MethodsCardiomyocytes were obtained from 1-day-old neonatal rat heart ventricles. To evaluate function of NRVMs cultured on ITO with electrical stimulation, the cell viability, change of cell morphology, immunochemistry using cardiac-specific antibodies, and gene expression were tested.ResultsDefined sarcomeric structure, cell enlargement, and increased distribution of NRVMs appeared in the presence of electrical stimulation. These characteristics were absent in NRVMs cultured under standard culture conditions. In addition, the expression levels of cardiomyocyte-specific and ion channel markers were higher in NRVMs seeded on ITO-coated dishes than in the control group at 14 days after seeding. ITO-coated dishes could effectively provide electrical cues to support the in vitro culture of NRVMs.ConclusionsThese results provide supporting evidence that electrical stimulation via ITO can be effectively used to maintain culture and enhance function of cardiomyocytes in vitro.

Histone methyltransferase Smyd1 regulates mitochondrial energetics in the heart

Smyd1, a muscle-specific histone methyltransferase, has established roles in skeletal and cardiac muscle development, but its role in the adult heart remains poorly understood. Our prior work demonstrated that cardiac-specific deletion of Smyd1 in adult mice (Smyd1-KO) leads to hypertrophy and heart failure. Here we show that down-regulation of mitochondrial energetics is an early event in these Smyd1-KO mice preceding the onset of structural abnormalities. This early impairment of mitochondrial energetics in Smyd1-KO mice is associated with a significant reduction in gene and protein expression of PGC-1α, PPARα, and RXRα, the master regulators of cardiac energetics. The effect of Smyd1 on PGC-1α was recapitulated in primary cultured rat ventricular myocytes, in which acute siRNA-mediated silencing of Smyd1 resulted in a greater than twofold decrease in PGC-1α expression without affecting that of PPARα or RXRα. In addition, enrichment of histone H3 lysine 4 trimethylation (a mark of gene activation) at the PGC-1α locus was markedly reduced in Smyd1-KO mice, and Smyd1-induced transcriptional activation of PGC-1α was confirmed by luciferase reporter assays. Functional confirmation of Smyd1's involvement showed an increase in mitochondrial respiration capacity induced by overexpression of Smyd1, which was abolished by siRNA-mediated PGC-1α knockdown. Conversely, overexpression of PGC-1α rescued transcript expression and mitochondrial respiration caused by silencing Smyd1 in cardiomyocytes. These findings provide functional evidence for a role of Smyd1, or any member of the Smyd family, in regulating cardiac energetics in the adult heart, which is mediated, at least in part, via modulating PGC-1α.

Electrophysiological Properties and Viability of Neonatal Rat Ventricular Myocyte Cultures with Inducible ChR2 Expression

Channelrhodopsin-2 (ChR2)-based optogenetic technique has been increasingly applied to cardiovascular research. However, the potential effects of ChR2 protein overexpression on cardiomyocytes are not completely understood. The present work aimed to examine how the doxycycline-inducible lentiviral-mediated ChR2 expression may affect cell viability and electrophysiological property of neonatal rat ventricular myocyte (NRVM) cultures. Primary NVRMs were infected with lentivirus containing ChR2 or YFP gene and subjected to cytotoxicity analysis. ChR2-expressing cultures were then paced electrically or optically with a blue light-emitting diode, with activation spread recorded simultaneously using optical mapping. Results showed that ChR2 could be readily transduced to NRVMs by the doxycycline-inducible lentiviral system; however, high-level ChR2 (but not YFP) expression was associated with substantial cytotoxicity, which hindered optical pacing. Application of bromodeoxyuridine significantly reduced cell damage, allowing stimulation with light. Simultaneous optical Vm mapping showed that conduction velocity, action potential duration, and dVm/dtmax were similar in ChR2-expressing and control cultures. Finally, the ChR2-expressing cultures could be optically paced at multiple sites, with significantly reduced overall activation time. In summary, we demonstrated that inducible lentiviral-mediated ChR2 overexpression might cause cytotoxicity in NRVM cultures, which could be alleviated without impairing electrophysiological function, allowing simultaneous optical pacing and Vm mapping.

Abstract 249: The Epigenetically Modified Pro-inflammatory Cardiac Fibroblast Drives Cardiomyocyte Dysfunction in Pulmonary Hypertension-induced Right Ventricular Pressure Overload

Right ventricular (RV) dysfunction is a strong predictor of survival in a variety of clinical contexts including pulmonary hypertension (PH). Thus elucidating mechanisms that contribute to RV dysfunction is of great importance. Persistent inflammation, a central component of PH-induced RV dysfunction, is regulated in part by cardiac fibroblasts (Cfib). We hypothesized that persistent changes in the Cfib pro-inflammatory phenotype may be mediated through epigenetic processes such as DNA methylation and that secretion of inflammatory signals from activated Cfib drives cardiomyocyte dysfunction and contributes to myofibrillar disarray. Therefore the purpose of this investigation was to assess epigenetic changes to pro-inflammatory Cfib and the contribution of the Cfib secretome on myocyte function. We approached this question using a large animal model with significant resonance with human disease- the neonatal calf exposed to hypobaric hypoxia (HH). HH caused RV Cfib global DNA hypomethylation. RNA-seq identified 2115 genes significantly changed in HH including 193 transcriptional regulators and 21 genes involved in DNA methylation. Further, we identified 105 inflammatory genes with enrichment of IL-1β as a central node of inflammatory signaling. Mass spec profiling of conditioned media (CM) from Cfib isolated from the PH calf (PH-CM) also confirmed significant pro-inflammatory protein secretion. We then examined the effects of PH-CM on adult rat ventricular myocytes (ARVM) in culture. Brief exposure to PH-CM caused marked dedifferentiation of ARVM to a neonatal-like phenotype that exhibited spontaneous contractile behavior. This response was not seen in ARVM exposed to CM from Cfib from the normoxic RV. These data suggest the combination of pressure overload and hypoxia causes epigenetic reprogramming of pro-inflammatory Cfib, signaling a phenotypic transformation in a population of myocytes which likely contributes to RV dysfunction. Therapies that target this inflammatory process have the potential to prevent RV dysfunction.

Mutual inhibition of insulin signaling and PHLPP-1 determines cardioprotective efficiency of Akt in aged heart.

Insulin protects cardiomyocytes from myocardial ischemia/reperfusion (MI/R) injury through activating Akt. However, phosphatase PHLPP-1 (PH domain leucine-rich repeat protein phosphatase-1) dephosphorylates and inactivates Akt. The balanced competitive interaction of insulin and PHLPP-1 has not been directly examined. In this study, we have identified the effect of mutual inhibition of insulin signaling and PHLPP-1 on the cardioprotective efficiency of Akt in aged heart. Young (3 mon) and aged (20 mon) Sprague Dawley (SD) rats were subjected to MI/R in vivo. The PHLPP-1 level was higher in aged vs. young hearts at base. But, insulin treatment failed to decrease PHLPP-1 level during reperfusion in the aged hearts. Consequently, the cardioprotection of insulin-induced Akt activation was impaired in aged hearts, resulting in more susceptible to MI/R injury. In cultured rat ventricular myocytes, PHLPP-1 knockdown significantly enhanced insulin-induced Akt phosphorylation and reduced simulated hypoxia/reoxygenation-induced apoptosis. Contrary, PHLPP-1 overexpression terminated Akt phosphorylation and deteriorated myocytes apoptosis. Using in vivo aged animal models, we confirmed that cardiac PHLPP-1 knockdown or enhanced insulin sensitivity by exercise training dramatically increased insulin-induced Akt phosphorylation. Specifically, MI/R-induced cardiomyocyte apoptosis and infarct size were decreased and cardiac function was increased. More importantly, we found that insulin regulated the degradation of PHLPP-1 and insulin treatment could enhance the binding between PHLPP-1 and β-transducin repeat-containing protein (β-TrCP) to target for ubiquitin-dependent degradation. Altogether, we have identified a new mechanism by which insulin suppresses PHLPP-1 to enhance Akt activation. But, aged heart possesses lower insulin effectiveness and fails to decrease PHLPP-1 during MI/R, which subsequently limited Akt activity and cardioprotection. PHLPP-1 could be a promising therapeutic interventional target for elderly ischemic heart disease patients.

Cardiomyocyte Antihypertrophic Effect of Adipose Tissue Conditioned Medium from Rats and Its Abrogation by Obesity is Mediated by the Leptin to Adiponectin Ratio.

White adipocytes are known to function as endocrine organs by secreting a plethora of bioactive adipokines which can regulate cardiac function including the development of hypertrophy. We determined whether adipose tissue conditioned medium (ATCM) generated from the epididymal regions of normal rats can affect the hypertrophic response of cultured rat ventricular myocytes to endothelin-1 (ET-1) administration. Myocytes were treated with ET-1 (10 nM) for 24 hours in the absence or presence of increasing ATCM concentrations. ATCM supressed the hypertrophic response to ET-1 in a concentration-dependent manner, an effect enhanced by the leptin receptor antagonist and attenuated by an antibody against the adiponectin AdipoR1 receptor. Antihypertrophic effects were also observed with ATCM generated from perirenal-derived adipose tissue. However, this effect was absent in ATCM from adipose tissue harvested from corpulent JCR:LA-cp rats. Detailed analyses of adipokine content in ATCM from normal and corpulent rats revealed no differences in the majority of products assayed, although a significant increase in leptin concentrations concomitant with decreased adiponectin levels was observed, resulting in a 11 fold increase in the leptin to adiponectin ratio in ATCM from JCR:LA-cp. The antihypertrophic effect of ATCM was associated with increased phosphorylation of AMP-activated protein kinase (AMPK), an effect abrogated by the AdipoR1 antibody. Moreover, the antihypertrophic effect of ATCM was mimicked by an AMPK activator. There was no effect of ET-1 on mitogen-activated protein kinase (MAPK) activities 24 hour after its addition either in the presence or absence of ATCM. Our study suggests that adipose tissue from healthy subjects exerts antihypertrophic effects via an adiponectin–dependent pathway which is impaired in obesity, most likely due to adipocyte remodelling resulting in enhanced leptin and reduced adiponectin levels.

Abstract 38: Simultaneous Optical Pacing and Optical Voltage Mapping in Optogenetic Neonatal Rat Ventricular Myocyte Cultures

Optogenetics is an emerging technology allowing remote and precise control of cell activity in living tissues. Despite its rapid advancements, application of this innovative technology to cardiovascular research is still limited, in part due to shortage of optogenetic cardiac tissue models and compatible imaging methods. The present study aimed to develop an optogenetic culture model using neonatal rat ventricular myocytes (NRVM) expressing light-gated Channelrhodopsin-2 (ChR2) and characterize activation spread during optical stimulation using optical mapping of membrane potential (Vm). Primary NVRM cultures were infected with lentivirus containing ChR2 gene. Cultures were paced electrically or optically with pulses of blue (470 nm) LED light. Activation spread was simultaneously mapped using Vm-sensitive dye (RH-237) and a photodiode mapping system. Results showed that ChR2 could be readily transduced to NRVMs by the lentiviral method; however, high-level ChR2 expression was associated with substantial cell toxicity. Lower ChR2 expression, achieved by administration of bromodeoxyuridine, had minor effects on cell morphology and function while allowing optical pacing at frequencies of 0.5-3 Hz. Simultaneous Vm mapping showed that conduction velocity, APD80, and dV/dtmax were similar in optogenetic and control cultures. Finally, the optogenetic cultures could be optically paced at multiple sites, leading to significantly reduced overall activation time. In summary, we demonstrated that ChR2 expression can cause cell toxicity in NRVM cultures but the toxicity can be mitigated allowing optical pacing and simultaneous optical activation mapping without significant impairment of electrophysiological function. This optogenetic cardiac culture model expands the availability of optogenetic tools for cardiac research.

Actin dynamics is rapidly regulated by the PTEN and PIP2 signaling pathways leading to myocyte hypertrophy.

Mature cardiac myocytes are terminally differentiated, and the heart has limited capacity to replace lost myocytes. Thus adaptation of myocyte size plays an important role in the determination of cardiac function. The hypothesis tested is that regulation of the dynamic exchange of actin leads to cardiac hypertrophy. ANG II was used as a hypertrophic stimulant in mouse heart and neonatal rat ventricular myocytes (NRVMs) in culture for assessment of a mechanism for regulation of actin dynamics by phosphatidylinositol 4,5-bisphosphate (PIP2). Actin dynamics in NRVMs rapidly increased in a PIP2-dependent manner, measured by imaging and fluorescence recovery after photobleaching (FRAP). A significant increase in PIP2 levels was found by immunoblotting in both adult mouse heart tissue and cultured NRVMs. Inhibition of phosphatase and tensin homolog (PTEN) in NRVMs markedly blunted ANG II-induced increases in actin dynamics, the PIP2 level, and cell size. Furthermore, PTEN activity was dramatically upregulated in ANG II-treated NRVMs but downregulated when PTEN inhibitors were used. The time course of the rise in the PIP2 level was inversely related to the fall in the PIP3 level, which was significant by 30 min in ANG II-treated NRVMs. However, significant translocation of PTEN to the plasma membrane occurred by 10 min, suggesting a crucial initial step for PTEN for the cellular responses to ANG II. In conclusion, PTEN and PIP2 signaling may play an important role in myocyte hypertrophy by the regulation of actin filament dynamics, which is induced by ANG II stimulation.

Abstract 299: Pulmonary Hypertension Activates Cardiac Fibroblasts To Release Paracrine Factors Regulating Cardiac Myocyte Dedifferentiation

In virtually all models of heart failure, prognosis is ultimately determined by right ventricular (RV) function. Thus understanding the unique cellular mechanisms that contribute to RV dysfunction is of critical importance. We have approached this question by studying a large animal model that has significant resonance with human disease: exposure of the neonatal calf to hypobaric hypoxia. This model demonstrates rapid onset of severe pulmonary hypertension (PH) and significant RV dysfunction. Strikingly, the RV of these animals shows a chamber-specific increase in markers of inflammation and local infiltration of activated pro-remodeling macrophages that are proportionate to the rise in PA pressure. In order to test the hypothesis that this inflammatory milieu directly contributes to myocyte dysfunction, we examined the effects of conditioned media (CM) from cardiac fibroblasts isolated from the PH calf on normal adult rat ventricular myocytes (ARVM) in culture. Even brief exposure (<2 days) to RV-CM results in rapid and marked dedifferentiation of ARVM to a neonatal-like phenotype that exhibits spontaneous contractile behavior. Dedifferentiated cells maintain viability for over 30 days with continued expression of cardiomyocyte proteins including TnI, sarcomeric actinin, and desmin. This response is not seen in ARVM exposed to CM from fibroblasts collected from the hypoxic LV or from the normoxic RV. We have characterized the factor(s) that are present in the RV-CM and have established that the active components are proteins with a mol wt >30 kDa and that inflammatory cytokines such as IL-18 are necessary but not sufficient to effect this phenotypic change. These data suggest that local and perhaps focal inflammation in the RV, induced by the combination of pressure overload and hypoxia, has the capacity to signal a phenotypic transformation in a population of RV cardiocytes, which likely contributes to chamber specific dysfunction. Therapies that target and interrupt this inflammatory process either by preventing in-migration of inflammatory cells or by blocking cell-cell signaling have the potential to prevent RV dysfunction.

Acetylation and Phosphorylation Post-Translational Modifications of the CapZ β1 Subunit Regulate FRAP Dynamics Leading to Myocyte Hypertrophy

The mechanism by which more thin filaments are built during cardiac hypertrophy is not fully understood. Very rapid increases the dynamics both actin and the actin capping protein (CapZ) following mechanical flexing suggest that a post-translational regulation is the underlying mechanism. Neonatal rat ventricular myocytes in culture were stimulated to hypertrophy by a neurohormone (10μM phenylephrine, PE, for 24 hr). CapZ dynamics were analyzed by fluorescence recovery after photobleaching (FRAP) using CapZβ1-GFP. After PE treatment, CapZ dynamics increased above resting controls by ∼3.17 fold (p=0.0004). Post-translational modifications of CapZ were analyzed by 2D gel electrophoresis. After PE treatment, 2D spots of CapZβ1-GFP have an increased negative shift, suggesting that post-translational modification of CapZ is up regulated. To identify the types of post-translational modifications, 2D western blotting and mass spectrometry (MS) was applied. Increased post-translational spots included the acetylation of K199 and phosphorylation of S204, which are both close to the actin-binding region of CapZ. To test whether CapZ acetylation was mediated by HDAC3, located at the Z-disc of myocytes, the class I HDAC inhibitor (5μM trichostatin A / 5hr) and HDAC3 activator (10μM theophylline / 24hr) were applied. CapZ dynamics with trichostatin A increased by four-fold (p=0.01), and the effect of PE on CapZ dynamics was blunted by theophylline (p=0.09). Thus, the increased sarcomere remodeling during cardiac hypertrophy may be induced via altered acetylation of CapZ, which is mediated by HDAC3. In addition to MS identification of phosphorylation sites, post-translational modifications were reduced by dominant negative PKCɛ, suggesting a regulatory role. Together the acetyl and phospho posttranslational modifications of CapZ reduce the capping property and may increase thin filament assembly. NIH HL62426 (BR) and AHA 12PRE12050371 (Y-H L).

Hydrogen Peroxide–Mediated SERCA Cysteine 674 Oxidation Contributes to Impaired Cardiac Myocyte Relaxation in Senescent Mouse Heart

BackgroundA hallmark of aging of the cardiac myocyte is impaired sarcoplasmic reticulum (SR) calcium uptake and relaxation due to decreased SR calcium ATPase (SERCA) activity. We tested the hypothesis that H2O2‐mediated oxidation of SERCA contributes to impaired myocyte relaxation in aging.Methods and ResultsYoung (5‐month‐old) and senescent (21‐month‐old) FVB wild‐type (WT) or transgenic mice with myocyte‐specific overexpression of catalase were studied. In senescent mice, myocyte‐specific overexpression of catalase (1) prevented oxidative modification of SERCA as evidenced by sulfonation at Cys674, (2) preserved SERCA activity, (3) corrected impaired calcium handling and relaxation in isolated cardiac myocytes, and (4) prevented impaired left ventricular relaxation and diastolic dysfunction. Nitroxyl, which activates SERCA via S‐glutathiolation at Cys674, failed to activate SERCA in freshly isolated ventricular myocytes from senescent mice. Finally, in adult rat ventricular myocytes in primary culture, adenoviral overexpression of SERCA in which Cys674 is mutated to serine partially preserved SERCA activity during exposure to H2O2.ConclusionOxidative modification of SERCA at Cys674 contributes to decreased SERCA activity and impaired myocyte relaxation in the senescent heart. Strategies to decrease oxidant levels and/or protect target proteins such as SERCA may be of value to preserve diastolic function in the aging heart.

Adiponectin inhibits leptin‐induced cardiomyocyte hypertrophy by attenuation of calcineurin/NFAT activation

Leptin and adiponectin are adipokines which exert opposite effects on cardiac pathology. Leptin induces cardiomyocyte hypertrophy and hyperleptinemia has been shown to be a risk factor for developing heart failure. In contrast adiponectin is antihypertrophic and reduced plasma adiponectin levels constitute a cardiovascular risk factor. In the present study we determined whether adiponectin can modify leptin‐induced hypertrophy in cultured rat ventricular myocytes. Leptin (3.1 nM) treatment for 24 h produced a robust hypertrophy as evidenced by significantly increased cell surface area, α‐skeletal actin gene expression and rate of protein synthesis. These effects were associated with significantly increased calcineurin activity and import of the transcriptional factor NFAT3 into nuclei. The leptin–induced calcineurin activation was paralleled by increased intracellular Na+ and Ca2+ concentrations. The pro‐hypertrophic effect of leptin was completely prevented in myocytes co‐treated with adiponectin (0.5 μM). These effects were associated with attenuation of leptin‐induced elevations in intracellular Na+ and Ca2+ concentrations, calcineurin activation and NFAT3 nuclear translocation. Thus, adiponectin may represent an endogenous factor mitigating the pro‐hypertrophic effect of leptin by preventing calcineurin activation. Supported by the Canadian Institutes of Health Research.

CapZ and actin capping dynamics increase in myocytes after a bout of exercise and abates in hours after stimulation ends

The time course of the response and recovery after acute activity seen in exercise is not well understood. The goal of this work is to address how proteins of the thin filament (actin and its capping protein CapZ) are changed by 1 h of mechanical stimulation and return to baseline over time. Neonatal rat ventricular myocytes in culture were subjected to cyclic 10% strain at 1 Hz for 1 h to mimic increased mechanical loading during exercise. CapZ and actin dynamics were analyzed by fluorescence recovery after photobleaching (FRAP) using CapZβ1-GFP, actin-GFP, or actin-RFP. After cyclic strain, CapZ dynamics increased above resting controls and abated 2-3 h after cessation of the cyclic strain. Similarly, actin dynamics initially increased and abated 1.5-2 h after the end of stimulation. Neurohormonal hypertrophic stimulation by phenylephrine or norepinephrine treatments also elevated actin dynamics but required a much longer time of treatment (24-48 h) to be detectable. The actin capping mechanism was explored by use of expression of CapZβ1 with a COOH-terminal deletion (CapZβ1ΔC). Increased dynamics of actin seen with CapZβ1ΔC was similar to the response to cyclic strain. Thus it is possible that mechanical stimulation alters the dynamics for CapZ capping of the actin filament through the CapZβ1 COOH terminus, known as the β tentacle, thereby remodeling sarcomeres in cardiac myocytes. This adaptive mechanism, which is probably regulating thin-filament addition, declines a few hours after the end of a bout of exercise.

Abstract 75: Insulin Modulates Cardioprotective Akt Signaling During Ischemia/Reperfusion Attenuating PHLPP-1 Phosphatase

Introduction The recently discovered phosphatase PHLPP-1 directly dephosphorylates and inactivates Akt. Whether the suppression of Akt by PHLPP-1 may lead to myocardial ischemia intolerance in aged heart, has never been previously studied. Moreover, we unveiled that the cardioprotective effects of insulin in limiting ischemia/reperfusion(I/R) injury were Akt activation dependent. However,the role of insulin,regulating the endogenous negative regulator of Akt activity, remains largely unknown. In this study, an important role for insulin in intensifying Akt activity through attenuating PHLPP-1 and ameliorating myocardial injury following I/R in the senescent heart has been described. Methods and Results Young (3 M) and aged (20 M) rats were subjected to I (20 min) / R (4 h) in vivo . Insulin treatment during reperfusion significantly increased Akt phosphorylation(2.32 times) response to I/R and decreased the infarct size by 18±3%. Simultaneously, insulin treatment significantly suppressed the PHLPP-1 level by 65±5%. Insulin-induced Akt activation was impaired and the myocardial infarct was larger in I/R aged hearts compared with the young ones. The effect of Insulin attenuating PHLPP-1 to intensify Akt activity was impaired in aged ischemic hearts as compared to young ischemic hearts. In cultured rat ventricular myocytes, the inhibitory effects of insulin on cardiac PHLPP-1 were abolished by the proteasome inhibitor MG132. PHLPP-1 knockdown by small interfering RNA significantly enhances insulin-induced Akt phosphorylation and reduced simulated hypoxia/reoxygenation induced apoptosis. Moreover, swimming training (6 w) incresed insulin sensitivity in the senescent heart. The inhibitory effect of insulin on cardiac PHLPP-1 was partially reversed accompanying enhanced Akt phosphorylation. The tolerance of the aged heart to ischemic damage is also enhanced. Conclusions We conclude that PHLPP-1 may contribute to the aging-associated ischemic intolerance. Insulin acts as a crucial cardioprotective regulator by inhibiting physiological function of PHLPP-1 and thus enchancing Akt activity. For the first time,our results also indicated a new cardioprotective mechanism of insulin modulating ubiquitin-proteasome pathway of PHLPP-1.

Endothelin Receptor Overexpression Alters Diastolic Function in Cultured Rat Ventricular Myocytes

The endothelin (ET) signaling pathway controls many physiological processes in myocardium and often becomes upregulated in heart diseases. The aim of the present study was to investigate the effects of ET receptor upregulation on the contractile function of adult ventricular myocytes. Primary cultured adult rat ventricular myocytes were used as a model system of ET receptor overexpression in the heart. Endothelin receptor type A (ETA) or type B (ETB) was overexpressed by Adenoviral infection, and the twitch responses of infected ventricular myocytes were measured after ET-1 stimulation. Overexpression of ETA exaggerated positive inotropic effect (PIE) and diastolic shortening of ET-1, and induced a new twitch response including twitch broadening. On the contrary, overexpression of ETB increased PIE of ET-1, but did not affect other two twitch responses. Control myocytes expressing endogenous receptors showed a parallel increase in twitch amplitude and systolic Ca2+ in response to ET-1. However, intracellular Ca2+ did not change in proportion to the changes in contractility in myocytes overexpressing ETA. Overexpression of ETA enhanced both systolic and diastolic contractility without parallel changes in Ca2+. Differential regulation of this nature indicates that upregulation of ETA may contribute to diastolic myocardial dysfunction by selectively targeting myofi lament proteins that regulate resting cell length, twitch duration and responsiveness to prevailing Ca2+.

Protective Role of Cardiac CFTR Activation Upon Early Reperfusion Against Myocardial Infarction

Background: The cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) was shown to be activated by β-adrenergic or purinergic stimulation and involved in cell volume regulation after osmotic swelling. Also, cardiac CFTR was reported to be essential in the mechanism by which ischemic preconditioning protects against ischemia/reperfusion(I/ R)-induced injury of the heart. Here, we explored the possibility that activation of cardiac CFTR can provide protection against I/R-induced myocardial infarction, even after ischemic attack. Methods: The hearts of wild-type mice were subjected to 30- or 40-min left coronary artery occlusion followed by 2-h or 2-day reperfusion in vivo, and myocardial infarction was examined under a variety of conditions. Neonetal rat ventricular myocytes in primary culture were subjected to hypoxia/reoxygenation in vitro, and necrotic cell death was examined. Results: The infarct size was much greater in CFTR knockout mice than in wild-type mice. Intravenous infusion of a number of putative CFTR activators upon reperfusion prominently reduced the size of myocardial infarction in wild-type but not CFTR-deficient mice. This protective effect was abolished by co-administration of a CFTR inhibitor. CFTR activators ameliorated, in a manner sensitive to a CFTR inhibitor, release of myocardial-specific creatine kinase isoenzyme to the serum in mice subjected to I/R in vivo. Necrotic death of cultured neonatal rat ventricular myocytes subjected to hypoxia/reoxygenation in vitro was ameliorated by CFTR activators or CFTR gene overexpression but aggravated by a CFTR inhibitor or CFTR gene silencing. Conclusion: It is concluded that activation of endogenous myocardial CFTR upon early reperfusion is involved in protection against necrotic myocardial injury induced by I/R in vivo and in vitro. Cardiac CFTR may serve as a target accessible even after ischemic attack for pharmacotherapeutic intervention in I/R-induced myocardial infarction.

Functional and morphological preservation of adult ventricular myocytes in culture by sub-micromolar cytochalasin D supplement

In cardiac myocytes, cytochalasin D (CytoD) was reported to act as an actin disruptor and mechanical uncoupler. Using confocal and super-resolution STED microscopy, we show that CytoD preserves the actin filament architecture of adult rat ventricular myocytes in culture. Five hundred nanomolar CytoD was the optimal concentration to achieve both preservation of the T-tubular structure during culture periods of 3days and conservation of major functional characteristics such as action potentials, calcium transients and, importantly, the contractile properties of single myocytes. Therefore, we conclude that the addition of CytoD to the culture of adult cardiac myocytes can indeed be used to generate a solid single-cell model that preserves both morphology and function of freshly isolated cells. Moreover, we reveal a putative link between cytoskeletal and T-tubular remodeling. In the absence of CytoD, we observed a loss of T-tubules that led to significant dyssynchronous Ca2+-induced Ca2+ release (CICR), while in the presence of 0.5μM CytoD, T-tubules and homogeneous CICR were majorly preserved. Such data suggested a possible link between the actin cytoskeleton, T-tubules and synchronous, reliable excitation–contraction-coupling. Thus, T-tubular re-organization in cell culture sheds some additional light onto similar processes found during many cardiac diseases and might link cytoskeletal alterations to changes in subcellular Ca2+ signaling revealed under such pathophysiological conditions.

Cardiac Myocyte-specific Ablation of Follistatin-like 3 Attenuates Stress-induced Myocardial Hypertrophy

Transforming growth factor-β family cytokines have diverse actions in the maintenance of cardiac homeostasis. Follistatin-like 3 (Fstl3) is an extracellular regulator of certain TGF-β family members, including activin A. The aim of this study was to examine the role of Fstl3 in cardiac hypertrophy. Cardiac myocyte-specific Fstl3 knock-out (KO) mice and control mice were subjected to pressure overload induced by transverse aortic constriction (TAC). Cardiac hypertrophy was assessed by echocardiography and histological and biochemical methods. KO mice showed reduced cardiac hypertrophy, pulmonary congestion, concentric LV wall thickness, LV dilatation, and LV systolic dysfunction after TAC compared with control mice. KO mice displayed attenuated increases in cardiomyocyte cell surface area and interstitial fibrosis following pressure overload. Although activin A was similarly up-regulated in KO and control mice after TAC, a significant increase in Smad2 phosphorylation only occurred in KO mice. Knockdown of Fstl3 in cultured cardiomyocytes inhibited PE-induced cardiac hypertrophy. Conversely, adenovirus-mediated Fstl3 overexpression blocked the inhibitory action of activin A on hypertrophy and Smad2 activation. Transduction with Smad7, a negative regulator of Smad2 signaling, blocked the antihypertrophic actions of activin A stimulation or Fstl3 ablation. These findings identify Fstl3 as a stress-induced regulator of hypertrophy that controls myocyte size via regulation of Smad signaling.